Directed repair or replacement of chromosomal gene sequences with cloned wild type or mutant DNA, also known as gene targeting, remains a technically challenging task. Although methods have been described for gene targeting in bacteria, most require specialized skills or material and often are time-consuming and laborious in practice. Linear DNA molecules possessing chi (khi) sites (5'-GCTGGTGG-3') participate in gene targeting replacement reactions through homologous recombination with the bacterial chromosome at much higher frequencies than DNAs lacking them. This project's goal is to develop a novel set of cloning vectors for gene targeting and gene knockout in E. coli and related enteric bacteria. Cloning vectors with multiple khi sites will be constructed and functionally tested for stimulation of gene targeting in vivo. Parameters to be evaluated address both the nature of the targeting DNA molecule and any influence that differ chromosomal target sites may have. A kit then will be developed enabling scientists to quickly and easily carry out gene targeting reactions, and should find widespread utility, for example, in mutating virulence genes from pathogenic bacteria. Longer term goals will be to apply this strategy to directed gene replacements in more distantly-related bacteria and eukaryotic systems. PROPOSED COMMERCIAL APPLICATION The potential commercial applications from this research are widespread, ranging from basic research through commercial uses including construction of stable bacterial strains for enzyme and pharmaceutical production to development of safer vaccines through the knockout of virulence genes. Moreover, it would be of high efficiency, the first kit of its kind, and could very well become the standard for bacterial gene targeting applications.